Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

Search Algorithms as a Framework for the Optimization of Drug Combinations

Combination therapies are often needed for effective clinical outcomes in the management of complex diseases, but presently they are generally based on empirical clinical experience. Here we suggest a novel application of search algorithms—originally developed for digital communication—modified to optimize combinations of therapeutic interventions. In biological experiments measuring the restoration of the decline with age in heart function and exercise capacity in Drosophila melanogaster, we found that search algorithms correctly identified optimal combinations of four drugs using only one-third of the tests performed in a fully factorial search. In experiments identifying combinations of three doses of up to six drugs for selective killing of human cancer cells, search algorithms resulted in a highly significant enrichment of selective combinations compared with random searches. In simulations using a network model of cell death, we found that the search algorithms identified the optimal combinations of 6–9 interventions in 80–90% of tests, compared with 15–30% for an equivalent random search. These findings suggest that modified search algorithms from information theory have the potential to enhance the discovery of novel therapeutic drug combinations. This report also helps to frame a biomedical problem that will benefit from an interdisciplinary effort and suggests a general strategy for its solution.     

The upscaling of transpiration from individual trees to areal transpiration in tree belts

Sap flow measurements have long been used to measure transpiration in individual trees and there exist some well established methods for upscaling individual tree volumetric transpiration to areal transpiration in plantation and forest plots. However, where edge effects are significant, such as in tree belts, the area the volumetric transpiration is to be projected upon is unknown. This paper provides a methodology for estimating the area that a tree belt hydrologically occupies by using either measurements of tree root density or soil moisture distribution. An application of the proposed methodology shows that simply assuming that the area of the tree belt is the crown projected area could lead to an overestimation of the areal transpiration of 100%. The online version of the original article can be found at .     

Structure and growth rates of the high-latitude coral: <Emphasis Type="Italic">Plesiastrea versipora</Emphasis>

The high-latitude coral species Plesiastrea versipora was investigated to identify growth rates in colonies over 1 m in diameter. Six colonies from two temperate gulfs (latitudes of 33°–35°S) in Southern Australia were examined using X-ray, luminescence and ²³⁸U/²³⁰Th dating techniques. Annual density bands were present in each coral but varied in width and definition, suggesting different linear extension and calcification rates. Differences in density band width were observed at the local scale (amongst colonies on the same reef) and regional scales (between the two gulfs). Extension rates of the P. versipora colonies examined in this study varied between 1.2 and 7 mm per year, which are amongst the slowest growth rates reported for hermatypic corals. As only one of the six P. versipora colonies had obvious luminescent banding, we conclude that luminescent banding is not an accurate chronological marker in this species of temperate water coral. Coral age estimates derived from counting density bands in X-radiographs ranged from 90 to 320 years for the six colonies studied. U-Th ages from the same colonies determined by high-precision multi-collector inductively coupled plasma mass spectrometer established radiometric ages between 105 and 381 years. The chronological variation in absolute ages between the two techniques varied between 2 and 19% in different colonies, with the lowest growth rates (~1 mm) displaying the greatest variation between density band age and radiometric U-Th age. This result implies that the age of P. versipora and other slow-growing corals cannot be determined accurately from density bands alone. The outcome of this research demonstrates that P. versipora may be useful as a paleoclimate archive, recording several centuries in a single colony in high-latitude environments (corals found in latitudes greater than 30° in either hemisphere), where other well-established coral climate archives, such as Porites, do not occur.     

The Role of FilGAP-Filamin A Interactions in Mechanoprotection

Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells. Suppression of force-induced lamella formation was mediated by repeat 23 of FLNa, which also binds FilGAP, a recently discovered Rac GTPase-activating protein (GAP). We found that FilGAP is targeted to sites of force transfer by FLNa. This force-induced redistribution of FilGAP was essential for the suppression of Rac activity and lamellae formation in cells treated with tensile forces. Depletion of FilGAP by small interfering RNA, inhibition of FilGAP activity by dominant-negative mutation or deletion of its FLNa-binding domain, all resulted in a dramatic force-induced increase of the percentage of annexin-V–positive cells. FilGAP therefore plays a role in protecting cells against force-induced apoptosis, and this function is mediated by FLNa.     

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease,is expressed by specific cell lineages during mouse embryonic development and in adult tissues

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.     

Cellular senescence in osteoarthritis pathology

Cellular senescence is a state of stable proliferation arrest of cells. The senescence pathway has many beneficial effects and is seen to be activated in damaged/stressed cells, as well as during embryonic development and wound healing. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been implicated in the pathogenesis of many age‐related diseases. Osteoarthritis (OA), a severely debilitating chronic condition characterized by progressive tissue remodeling and loss of joint function, is the most prevalent disease of the synovial joints, and increasing age is the primary OA risk factor. The profile of inflammatory and catabolic mediators present during the pathogenesis of OA is strikingly similar to the secretory profile observed in ‘classical’ senescent cells. During OA, chondrocytes (the sole cell type present within articular cartilage) exhibit increased levels of various senescence markers, such as senescence‐associated beta‐galactosidase (SAβGal) activity, telomere attrition, and accumulation of p16ink4a. This suggests the hypothesis that senescence of cells within joint tissues may play a pathological role in the causation of OA. In this review, we discuss the mechanisms by which senescent cells may predispose synovial joints to the development and/or progression of OA, as well as touching upon various epigenetic alterations associated with both OA and senescence.     

A note on type II error under random effects misspecification in generalized linear mixed models

Litière, Alonso, and Molenberghs (2007, Biometrics, 63, 1038-1044) presented the results of simulation studies that they claimed showed that misspecification of the shape of the random effects distribution can produce marked increases in Type II error (decreases in power) of tests based on fits of generalized linear mixed models. However, the article contains a logical fallacy that invalidates this claim. We present logically correct simulation studies that demonstrate little increase in Type II error, consistent with the earlier work that shows little effect due to misspecification.